Dp72 image acquisition system

After sacrificing by asphyxiation, the intestinal tissues of mice were fixed with 4% formaldehyde, embedded in paraffin and sliced, baked at 60°C for 2 h, rinsed in xylene and gradient alcohol. The slices were placed in 0.01 mol/L citrate buffer (PH = 6.0) for antigen retrieval at 98°C using microwave for 20 min. The slices were further incubated with 3% hydrogen peroxide at room temperature for 10 min to eliminate endogenous peroxidase, blocked with 2% bovine serum albumin (BSA) at room temperature for 30 min, incubated with anti-NLRP3 and anti-Caspase-1 and monoclonal antibodies (Abcam, MA, USA) (dilution 1:300). After washing with for 3 times, the slices were incubated with proper secondary antibody (Goat anti rabbit IgG H + L) at 37°C for 15 min, incubated with peroxidase-labeled streptomycin (Maxim Biotechnology Company, Fuzhou, China) for 15 min and wash with PBS for 3 times (5 min each). The slices were further visualized with freshly prepared DAB solution (Dako, Glostrup, Denmark), counterstained with hematoxylin and mounted. For negative control, primary antibody was replaced by TBS. The Olympus-DP72 image acquisition system and the Olympus-BX51 upright microscope of the CRi Nauance multispectral imaging system (Cambridge Research and Instrumentation, MA, USA) were used to acquire pictures and for quantitative analysis.

Mouse intestinal tissue was fixed with 4% PFA, paraffin‐embedded and then sliced, baked at 60℃ for 2 h, treated with xylene and gradient alcohol. The slices were placed in 0.01 mol/L citrate buffer (PH = 6.0), heated at 98℃ in a microwave for 20 min for antigen retrieval, incubated with 3% hydrogen peroxide at room temperature for 10 min to eliminate endogenous peroxidase, blocked with 2% bovine serum albumin (BSA) at room temperature for 30 min, incubated with monoclonal antibodies against MD2, occludin and claudin‐1 (Abcam, dilution 1:300), washed with TBS for three times, added with proper secondary antibody at 37℃ for 15 min, incubated with peroxidase‐labelled streptomycin (Mexin Biotechnology Development Company) for 15 min, rinsed with PBS for three times (5 min each time), visualized with freshly prepared DAB solution (Dako), counterstained with haematoxylin and finally mounted. For negative control, primary antibody was replaced by TBS. The Olympus‐DP72 image acquisition system and the Olympus‐BX51 upright microscope of the CRi Nuance multispectral imaging system (Cambridge Research & Instrumentation) were used for image acquisition and quantitative analysis.